The present invention relates to a chelation complex comprising 225Actinium (225Ac) and 1,4,7,10,13,16-hexaazacyclooctodecane-N,Nxe2x80x2,Nxe2x80x3,Nxe2x80x2xe2x80x3,Nxe2x80x3xe2x80x3,Nxe2x80x2xe2x80x3xe2x80x3-hexaacetic acid (HEHA) (225Ac-HEHA), bifunctional HEHA, bifunctional HEHA-targeting agent, bifunctional 225Ac-HEHA, bifunctional 225Ac-HEHA-targeting agent, and methods of synthesis and use, such as in the context of radioimmunotherapy, decontamination and detoxification.
In the field of radioimmunotherapy, the radioisotope chosen is determined, at least in part, by the type of disease to be treated. The reason for this is that the type of particles emitted by a given radioisotope are directly related to tissue penetration and the ability of the isotope to kill cells (Boll et al., Radiochim. Acta 79: 87-91 (1997)). xcex2-emitters, like 90Y and 131I, which have a tissue range of several millimeters, have been used successfully to treat solid tumors (Boll et al. (1997), supra). However, a tissue range of several millimeters is not optimal for the treatment of single cells, small clusters of cells, micrometastatic disease, leukemias and lymphomas (Jurcic et al., In: Cancer Chemotherapy and Biological Response Modifiers Annual 17, Pinedo et al., eds., New York: Elsevier B. V. (1998), pp. 195-216; Falini et al., Cancer Surveys 30: 295-309 (1998)). xcex1-emitters, on the other hand, combine high cytotoxicity with a short tissue range, i.e., less than about 150 xcexc (Boll et al. (1997), supra). Alpha radiation can kill a cell with only one hit to the nucleus and will kill substantially any cell with 10 hits or less. Consequently, considerable effort has been expended in the development of the xcex1-emitters 212Bi (txc2xd=60 min) (Ruegg et al., Cancer Res. 50: 4221-4226 (1990)), 213Bi (txc2xd=45 min) (Geerlings et al., Nucl. Med. Comm. 14: 121-125 (1993)), and 211At (txc2xdxe2x80x947.2 hr) (Lambrecht et al., Radiochim. Acta 36: 443-440 (1985)). However, 212Bi, 213Bi and 211At suffer from disadvantages. The short half-life of 212Bi and 213Bi limit their application. The limited available of 211At, due to half-life and production constraints, limits its utility. Consequently, 225Ac, which is highly cytotoxic, has been proposed as an alternative xcex1-emitter to 212Bi, 213Bi and 211At for use in radioimmunotherapy.
225Ac decays through a chain of four xcex1 emissions and two xcex2 emissions to the stable isotope 209Bi, thereby releasing a large amount of energy (28 MeV) (Davis et al., Nucl. Med. Biol., accepted; Alleluia et al., In: Gmelin Handbook of Inorganic Chemistry, 8th ed., Kugler et al., eds., New York: Springer-Verlag (1981), pp. 181-193). Unfortunately, most of the 221Ac administered in a dose is deposited in the liver and bone (Beyer et al., Isotopenpraxis 26: 111-114 (1990)). Thus, numerous attempts have been made to reduce the toxicity of 225Ac through chelation with, for example, citrate (Beyer et al. (1990), supra), EDTMP (ethylenediaminetetramethylenephosphonic acid; Beyer et al., Nucl. Med. Bio. 24:367-372 (1997)), EDTA (ethylenediaminetetraacetic acid; Alleluia et al. (1981), supra) and CHXAxe2x80x3-DTPA (N[(R)-2-amino-3-(4-nitrophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-N,N,Nxe2x80x2,Nxe2x80x3,Nxe2x80x3-pentaacetic acid; Davis et al., supra). While these chelates reduce the liver dose somewhat, CHXAxe2x80x3-DTPA, which is the best 225Ac chelate to date, still has a maximum tolerated dose (MTD) of approximately 100 kBq in mice and higher doses of 225Ac-CHX-DTPA have resulted in 100% mouse mortality within eight days (Davis et al., supra).
Thus, while 225Ac is potentially useful in radioimmunotherapy, a suitable chelate is needed. Until now, a suitable chelate with sufficient in vivo stability had yet to be discovered. Certain bifunctional large cyclic chelate ligands are described in JP3-197468. However, there is no teaching or suggestion in JP3-197468 that the disclosed ligands would be useful to chelate 225Ac. Accordingly, it is an object of the present invention to provide chelated 225Ac. It is another object of the present invention to provide methods of synthesizing the chelate and related compounds. It is yet another object of the present invention to provide methods of using the chelate and related compounds. These and other objects, as well as additional advantages and inventive features, will become apparent from the detailed description provided herein.
The present invention provides an xcex1-particle-emitting radioisotope chelation complex comprising 225Actinium (225Ac) and 1,4,7,10,13,16-hexaazacyclooctodecane-N,Nxe2x80x2,Nxe2x80x3,Nxe2x80x2xe2x80x3,Nxe2x80x3xe2x80x3,Nxe2x80x2xe2x80x3xe2x80x3-hexaacetic acid (HEHA) (225Ac-HEHA). Also provided is a bifunctional HEHA which can chelate a radiosotope, in particular 225Ac, and can be attached to a targeting agent, such as a bifunctional HEHA having one of the following formulae: 
wherein R is CO2H, CONHRxe2x80x2, P(O)Rxe2x80x2OH or P(O) (ORxe2x80x2)CH, Rxe2x80x2 is H, a C1-C8 alkyl, phenyl or benzyl, wherein said phenyl or benzyl is unsubstituted or substituted, n is 1-6, X is NO2, NH2, NCS, NHC(O)CH2Z (in which Z is Cl, Br or I), or 
Other bifunctional HEHAs are set forth herein. A compound comprising the bifunctional HEHA conjugated to a targeting agent is also provided. Accordingly, further provided is a bifunctional 225Ac-HEHA complex comprising 225Ac complexed with the bifunctional HEHA described above as well as a compound comprising the bifunctional 225Ac HEHA complex conjugated to a targeting agent.
In view of the above, the present invention further provides a method of making HEHA. The method comprises preparing the free base of 1,4,7,10,13,16-hexaazacyclooctodecane under anhydrous conditions, azeotropically removing trace water with benzene, N-alkylating the macrocycle to produce the hexaester, saponifying the hexaester, and purifying HEHA. Preferably, the hexaester is produced by reacting the free base with Na2CO3 and tert-butyl bromoacetate in anhydrous CH3CN.
Still further provided is a method of making a bifunctional HEHA. The method comprises the preparation of a tert-butyloxycarbonyl protected iminodiacetic acid that is condensed with an amino acid ester. The resulting diester is then saponified with base, and after acidification, converted to a disuccinimidyl ester. This active diester is then reacted with an N-2-aminoethyl amide of para-nitrophenylalanine that introduces the latent bifunctionality aspect that will be unmasked. The protecting group is removed by treatment with acid, and the amide carbonyl functional groups are reduced via diborane. The resulting macrocyclic polyamine is isolated as the protonated salt. The free base is generated and then the free amines are alkylated to introduce protected R groups. The protected R groups are then deprotected. The nitro group is then hydrogenated to the aniline, which is then converted to an isothiocyanate, a haloacetamide or a maleimide for conjugation to a targeting agent. The method can further comprise the conjugation of a bifunctional HEHA to a targeting agent. Alternatively, the method comprises the preparation of a cyclic hexapeptide that comprises para-nitrophenylalanine or xcex5-protected lysine and the subsequent reduction of amide carbonyl functional groups. The resulting macrocyclic polyamine is isolated as the protonated salt. The free base is then generated and the free amines are alkylated to introduce protected R groups, which are subsequently deprotected. The nitro group is hydrogenated to the aniline and the aniline is converted to an isothiocyanate, a haloacetamide or a maleimide, any one of which can then be conjugated to a targeting agent.
In another embodiment, a method of treating disease is provided. The method comprises administering to a patient having disease a disease-treatment effective amount of a 225Ac-HEHA targeting agent as described above in which the targeting agent is specific for diseased cells.
In yet another embodiment, a method of treating cancer is also provided. The method comprises administering to a patient having cancer a cancer-treatment effective amount of a 225Ac-HEHA-targeting agent as described above in which the targeting agent is specific for the cancer to be treated. In a related embodiment, a method of treating a solid tumor is provided. The method comprises intratumorally administering to a patient having a tumor a tumor-treatment effective amount of 225Ac-HEHA or 225Ac-HEHA-targeting agent in which the targeting agent is specific for the tumor. Optionally, the method further comprises simultaneously or sequentially peritumorally administering to the patient HEHA in an amount effective to chelate any radioactive decay products from the compound.
In still yet another embodiment, a method of decontaminating a sample from 225Ac is provided. The method comprises contacting the sample with a decontaminating-effective amount of HEHA.
A further embodiment is a method of decontaminating a person who has been externally contaminated with 225Ac. The method comprises contacting the person with a decontaminating-effective amount of HEHA. Similarly, a method of detoxifying a person who has internalized 225Ac is provided. The method comprises administering to the person a detoxifying-effective amount of HEHA.
The present invention is predicated on the surprising and unexpected discovery that HEHA chelates 225Ac in such a manner as to provide sufficient in vivo stability to enable its use in the context of radioimmunotherapy and other contexts. Accordingly, in one embodiment, the present invention provides an xcex1-particle-emitting radioisotope chelation complex comprising 225Ac and HEHA. 225Ac-HEHA is highly desirable because it comprises 225Ac, which is a metal radioisotope with excellent cytotoxicity that forms a suitable complex with HEHA having a half-life (i.e., approximately 10.5 days, wherein from about 30 min to about 3 wks is preferred and from about 30 min to about 11 days is more preferred) and an emission quality that are characteristic of radiopharmaceuticals and a toxic radioactive decay chain that results in nonradioactive material as a final product.
In view of the above, the present invention further provides a bifunctional HEHA which can chelate a radiosotope, which is preferably 225Ac, and can attach to a targeting agent, such as described herein. HEHA can be rendered bifunctional in any suitable manner in accordance with methods known in the art (see, e.g., Wang, Chemistry of Protein Conjugation and Crosslinking, CRC Press, Boca Raton, Fla. (1991); Lundblad, Chemical Reagents for Protein Modification, CRC Press, Boca Raton, Fla. (1991)). Preferred bifunctional HEHAs include those of the following formulae: 
wherein R is selected from the group consisting of CO2H, CONHRxe2x80x2, P(O)Rxe2x80x2OH and P(O)(ORxe2x80x2)OH,
Rxe2x80x2 is selected from the group consisting of H, a C1-C8 alkyl, phenyl and benzyl, wherein phenyl and benzyl are substituted or unsubstituted,
n is 1-6 and,
X is selected from the group consisting of NO2, NH2, NCS, NHC(O)CH2Z (in which Z is selected from the group consisting of Cl, Br and I), and 
Preferably, R is CO2H and Rxe2x80x2 is H or CH3. When Rxe2x80x2 is phenyl or benzyl, phenyl or benzyl can be substituted with one or more substituents selected from the group consisting of a C1-C6 alkyl, a halogen, a C1-C6 alkoxy, a C1-C6 hydroxyl, and a C1-C6 poly-hydroxyl.
Other bifunctional HEHAs include those of formulae: 
wherein R, Rxe2x80x2, n and x are as defined above and R3 is selected from the group consisting of H, a C1-C6 alkyl, and benzyl.
Further in view of the above, the present invention provides a compound comprising the above-described bifunctional HEHA conjugated to a targeting agent. By xe2x80x9ctargeting agentxe2x80x9d is meant any means that enables specific interaction with a target. The targeting agent can bind to a defined population of cells, for example, through a receptor, a substrate, an antigenic determinant or another binding site on the target cell population.
Cell-surface molecules that are cancer specific antigens (or disease-specific antigens) and can serve as targets are known in the art.
Examples of cancer-specific, cell-surface molecules include placental alkaline phosphatase (testicular and ovarian cancer), pan carcinoma (small cell lung cancer), polymorphic epithelial mucin (ovarian cancer), prostate-specific membrane antigen, xcex1-fetoprotein, xcex2-lymphocyte surface antigen (B-cell lymphoma), truncated EGFR (gliomas), idiotypes (B-cell lymphoma), gp95/gp97 (melanoma), N-CAM (small cell lung carcinoma), cluster w4 (small cell lung carcinoma), cluster 5A (small cell carcinoma), cluster 6 (small cell lung carcinoma), PLAP (seminomas, ovarian cancer, and non-small cell lung cancer), CA-125 (lung and ovarian cancers), ESA (carcinoma), CD19, 22 or 37 (B-cell lymphoma), 250 kD proteoglycan (melanoma), P55 (breast cancer), TCR-IgH fusion (childhood T-cell leukemia), blood group A antigen in B or O type individual (gastric and colon tumors), and the like.
Examples of cancer-specific, cell-surface receptors include erbB-2, erbB-3, erbB-4, IL-2 (lymphoma and leukemia), IL-4 (lymphoma and leukemia), IL-6 (lymphoma and leukemia), MSH (melanoma), transferrin (gliomas), tumor vasculature integrins, and the like. Preferred cancer-specific, cell-surface receptors include erbB-2 and tumor vasculature integrins, such as CD11a, CD11b, CD11c, CD18, CD29, CD51, CD61, CD66d, CD66e, CD106, and CDw145.
The erbB-2 receptor has been found in breast, ovarian, gastric, salivary gland and adeno-carcinomas and in non-small cell carcinomas of the lung. Over-expression of the erbB-2 receptor on such cancers has been found to correlate with poor prognosis. In vitro studies strongly suggest that over-expression of erbB-2 may play an important role in tumor progression.
An example of a single-chain antibody scAb is that which binds c-erbB-2 (WO 93/16185). See, also, WO 93/21232 and H. Zola, Monoclonal Antibodies, BIOS Scientific Publishers, Oxfordshire, England (November 1994) antibody sequences that can be used to construct scAbs.
There are a number of antibodies to cancer-specific, cell-surface molecules and receptors that are known. C46 Ab (Amersham) and 85A12 Ab (Unipath) to carcino-embryonic antigen, H17E2 Ab (ICRF) to placental alkaline phosphatase, NR-LU-10 Ab (NeoRx Corp.) to pan carcinoma, HMFC1 Ab (ICRF) to polymorphic epithelial mucin, W14 Ab to B-human chorionic gonadotropin, RFB4 Ab (Royal Free Hospital) to B-lymphocyte surface antigen, A33 Ab (Genex) to human colon carcinoma, TA-99 Ab (Genex) to human melanoma, antibodies to c-erbB2 (JP 7309780, JP 8176200 and JP 7059588), and the like. ScAbs can be developed, based on such antibodies, using techniques known in the art (see, for example, Bind et al., Science 242: 423-426 (1988), and Whitlow et al., Methods 2(2): 97-105 (1991)).
Examples of binding domains include the EGF domain of xcex1-heregulin, xcex1-integrin domain, tumor vasculature peptide motifs. Alpha-heregulin is a ligand with affinity for breast cancer cells expressing the human epidermal growth factor receptors erbB-2, erbB-3 and erbB-4. Heregulin interacts indirectly with erbB-2 via heterodimerization with erbB-3 or erbB-4.
In general, there are a number of databases for ligands, binding domains and cell-surface molecules.
Examples of a targeting agent include an xe2x80x9cimmunological agent,xe2x80x9d which is used herein to refer to an antibody, such as a polyclonal antibody or a monoclonal antibody, an immunologically reactive fragment of an antibody, an engineered immunoprotein and the like, a protein (target is receptor, as substrate, or regulatory site on DNA or RNA), a peptide (target is receptor), a nucleic acid (target is complementary nucleic acid), a steroid (target is steroid receptor), and the like. Preferred targeting agents include an antibody or an iummunologically reactive fragment thereof, a peptide, e.g., bombesin, gastrin-releasing peptide, RGD peptide, substance P, neuromedin-B, neuromedin-C, somatostatin, octreotide analogues, and metenkephalin, and a hormone, e.g., estradiol, neurotensin, melanocyte stimulating hormone, follicle analogues stimulating hormone, leutenizing hormone, and human growth hormone. Other suitable targeting agents include serum proteins, fibrinolytic enzymes, and biological response modifiers, such as interleukin, interferon, erythropoietin, and colony-stimulating factor. Analogs of targeting agents that retain the ability to bind to a defined target also can be used. In addition, synthetic targeting agents can be designed, such as to fit a particular epitope. The targeting agent can include any linking group that can be used to join a targeting agent to, in the context of the present invention, a chelate. It will be evident to one skilled in the art that a variety of linking groups, including bifunctional reagents, can be used.
Accordingly, the present invention further provides a bifunctional 225Ac-HEHA comprising 225Ac complexed with a bifunctional HEHA as described above. Also, in this regard, the present invention provides a compound comprising a bifunctional 225Ac-HEHA conjugated to a targeting agent as described above.
Also provided by the present invention is a method of attaching HEHA, which is not bifunctional, to a protein. See, for example, the method set forth in Example 11. Accordingly, the present invention further provides a HEHA-protein compound.
In another embodiment of the present invention, a method of making HEHA is provided. The method comprises preparing the free base of the macrocycle 1,4,7,10,13,16-hexaazacyclooctodecane under anhydrous conditions, azeotropically removing trace water with benzene, N-alkylating the macrocycle to produce the hexaester, saponifying the hexaester, and purifying HEHA. Preferably, the hexaester is produced by reacting the free base with Na2CO3 and tert-butyl bromoacetate in anhydrous CH3CN. A preferred method is set forth in Example 1.
In the practice of the present invention, 225Ac can be chelated either before or after the chelator is conjugated to a targeting agent. The order chosen can take into account stability and other factors and is well within the ordinary skill in the art.
Methods of complexing metal ions with chelants are known and are within the level of ordinary skill in the art. A metal can be incorporated into a chelant moiety by one of three general methods, i.e., direct incorporation, template synthesis and/or transmetallation. Direct incorporation is preferred.
Generally, the metal is titrated from substoichiometric levels up to full incorporation, thus eliminating the need for dialysis and extensive chromatographic purification. In this manner, significant losses as well as dilution are avoided.
Generally, a water-soluble form of the metal, such as an inorganic salt, is dissolved in an appropriate volume of distilled, deionized water, preferably in a dilute acid medium having a pH of from about 1 to about 7 and most preferably at a pH of from about 4 to about 6. Ambient temperatures of about 20xc2x0 C. to 27xc2x0 C. or below (to just above freezing) can be readily employed with stirring for metal chelation. Any appropriate metal salt, either in solid form or in solution, can be contacted with the chelate, either free in solution or conjugated to a targeting agent, in order to form the chelated 225Ac. The pH of the mixture is raised slowly by addition of base, typically 0.1 M NaOH, until the donor groups of the polychelant are deprotonated, generally in the pH range of from about 5 to about 9, depending on the chelant moieties. Particular care must be taken to maintain the pH below 8 to avoid precipitation of the metal hydroxide. Preferred methods include those set forth in Examples 2 and 6.
A wide variety of metal salts can be employed including, for example, nitrates, iodides, chlorides, citrates, acetates and the like. The choice of an appropriate metal salt as well as the choice of a particularly appropriate chelate for any given metal is within the ordinary skill in the art. It will be apparent that the practice of this invention permits the processing of rather small quantities of metal and targeting agent to form metal chelate and metal chelate targeting agent conjugates. The amount of metal employed may be from trace amounts to amounts in excess of equimolar with the chelate.
In still yet another embodiment of the present invention, a method of making a bifunctional HEHA is provided. Preferred methods include those set forth in Example 3. The method comprises the preparation of a tert-butyloxycarbonyl protected iminodiacetic acid that is condensed with an amino acid ester. The resulting diester is then saponified with base, and after acidification, converted to a disuccinimidyl ester. This active diester is then reacted with an N-2-aminoethyl amide of para-nitrophenylalanine that introduces the latent bifunctionality aspect that will be unmasked. The protecting group is removed by treatment with acid, and the amide carbonyl functional groups are reduced via diborane. The resulting macrocyclic polyamine is isolated as the protonated salt. The free base is generated and then the free amines are alkylated to introduce protected R groups. The protected R groups are then deprotected. The nitro group is then hydrogenated to the aniline, which is then converted to an isothiocyanate, a haloacetamide or a maleimide for conjugation to a targeting agent.
The methods described are well-defined and understood and available to those skilled in the art. Choices of protecting groups, active esters, coupling reagents, and conjugation reactive groups are within the literature. See, for example, Bodanszky, Principles of Peptide Synthesis, 2nd Ed., Springer-Verlag, NY (1993); Green et al., Protective Groups in Organic Synthesis, 2nd Ed., John Wiley and Sons, Inc. (1991); Wong, Chemistry of Protein Conjugation and Crosslinking, CRC Press, Boca Raton, Fla. (1991); Lundblad, Chemical Reagents for Protein Modification, CRC Press, Boca Raton, Fla. (1991); and Magerstadt, Antibody Conjugates and Malignant Disease, CRC Press, Boca Raton, Fla. (1991).
Another route to prepare bifunctional HEHA reagents is via the preparation of the cyclic hexapeptide, wherein the amino acid components provide the latent bifunctionality aspect that will be unmasked, specifically an amino acid such as of para-nitrophenylalanine or an xcex5-protected lysine. The advantage to this method is the potential to introduce additional functional groups into the macrocyclic ring stereospecifically and thus tune the cavity size of the macrocycle. The ring is formed from the linear hexapeptide by cyclization with an activating and/or dehydrating reagent, for example DPPA (diphenylphosphoryl azide). The carbonyl functional groups are reduced via diborane. The free amines in the resulting macrocyclic polyamine are then alkylated to introduce protected R groups. The protected R groups are then deprotected. The nitro group is then hydrogenated to the aniline, which is then converted to an isothiocyanate, a haloacetamide or a maleimide for conjugation to a targeting agent. See, for example, Bodanszky (1993), supra; Green et al. (1991), supra; Wong (1991), supra; Lundblad (1991), supra; Magerstadt (1991), supra; and Aston et al., Tetrahedron Lett. 35:3687-3690 (1994).
If 225Ac-HEHA, wherein the HEHA is bifunctional, or the bifunctional HEHA is to be conjugated, 225Ac-HEHA or the bifunctional HEHA is mixed in aqueous solution with the desired targeting agent, such as an antibody, at a pH of from about 6 to about 11, most preferably at a pH of from about 7 to about 9.5. Desirably, the pH is adjusted with a buffered solution, such as a bicarbonate buffered solution. Once again, the choice of an appropriate buffer is within the ordinary skill in the art. The temperature of the solution can range from just above freezing to the temperature at which the chelate becomes unstable or the protein denatures. Often temperatures above 37xc2x0 C. tend to denature proteins. Generally, chelate and targeting agent are mixed in a molar ratio of greater than 1:1 and less than 100:1 depending on protein concentration. Ratios of about 2:1 to about 4:1 are preferred, but the choice of reaction conditions is within the ordinary skill in the art.
Accordingly, in still yet another embodiment of the present invention, a method of attaching a bifunctional HEHA to a targeting agent is provided. The method comprises the conjugation of a bifunctional HEHA to a targeting agent or vector. Preferred methods include those set forth in Example 4. In one particular embodiment, the targeting agent was a monoclonal antibody. The antibody was prepared for conjugation by transfer of the antibody by dialysis to the conjugation buffer (bicarbonate, 0.5 M EDTA, pH=8.0). After dialysis, the concentration was determined spectrophotometrically. To the antibody solution, a solution of a bifunctional HEHA in water was added. Typical initial ratios used to achieve the desired final chelate/protein ratio of 1-2 were a 10-fold initial excess of ligand for whole antibodies and a 10-15-fold initial excess of ligand for antibody fragments. After the conjugation reaction was completed (18 hr), the reaction mixture was purified by centrifugation filtration. The buffer was changed to ammonium acetate buffer (0.15 M, pH=7.0) by centrifugation filtration. Alternately, dialysis against 1 l of 0.15 M NH4OAc for a minimum of 6 hr and changing buffer for a total of 4 times provided the conjugate ready for radiolabeling.
The conjugates can be used as such with appropriate pH adjustment, if needed. Alternatively, if it is desired to purify the conjugate from unconjugated chelate or products of any side reactions, the product can be purified. A variety of standard purification techniques known in the art can be used, including column chromatography and high performance liquid chromatography (HPLC).
225Ac-HEHA and conjugates thereof can be administered in vivo in the form of a composition, e.g., a pharmaceutical composition, comprising a carrier, e.g., pharmaceutically acceptable carrier. A biologically acceptable, normal saline solution can be appropriately employed. The carrier can include a minor amount of a carrier protein, such as human serum albumin, for example, to stabilize the targeting agent. Stabilizers, antioxidants, osmolality adjusting agents, buffers, pH adjusting agents, etc., can be included in the composition. The composition can be in the form of a solution, suspension or dispersion. Suitable additives include, for example, physiologically biocompatible buffers, additions of chelants or calcium chelate complexes, or optionally, additions of calcium or sodium salts.
Parenterally administrable forms, e.g., intravenous forms, should be sterile and free from physiologically unacceptable agents and should have low osmolality to minimize irritation or other adverse effects upon administration. Suitable vehicles include aqueous vehicles customarily used for administering parenteral solutions, such as sodium chloride injection, Ringer""s injection, dextrose injection, dextrose and sodium chloride injection. Lactated Ringer""s injection and other solutions are as described in Remington""s Pharmaceutical Sciences, 15th ed., Easton: Mack Publishing Co. (1975). The solutions can contain preservatives, antimicrobial agents, buffers and antioxidants conventionally used for parenteral solutions, excipients and other additives that are compatible with the chelates and will not interfere with the manufacture, storage or use of products.
The concentration of 225Ac-HEHA or conjugate thereof in a composition will be a matter of choice. Levels of 0.5 mg/ml are readily attainable but the concentration may vary considerably depending upon the specifics of any given application. Appropriate concentrations of biologically active materials in a carrier are routinely determined in the art.
The effective dose (referred to herein as xe2x80x9cdisease-treatment effective amount,xe2x80x9d xe2x80x9ccancer-treatment effective amount,xe2x80x9d xe2x80x9ctumor-treatment effective amount,xe2x80x9d xe2x80x9cdecontaminating effective amountxe2x80x9d and xe2x80x9cdetoxifying-effective amountxe2x80x9d) of HEHA, 225Ac-HEHA or conjugate of either of the foregoing to be utilized for any application will also depend upon the particulars of that application. In treating tumors in the context of radioimmunotherapy, for example, the dose will depend, inter alia, upon tumor burden, accessibility, route of administration, administration of other active agents, and the like. Generally, a therapeutically effective dose is from about 20 mCi to about 300 mCi.
HEHA and 225Ac-HEHA-targeting agent can be administered in accordance with the present inventive methods by any suitable route. Such routes include intravenous, intraperitoneal, and the like, depending on the site of contamination with 224Ac or the disease or cancer to be treated, respectively, the location of the contaminated/diseased/cancerous cells, the extent of contamination/disease/cancer, and other factors. The determination of the appropriate route(s) of administration for a given application is within the ordinary skill in the art. In the treatment of prostate cancer, for example, transurethral delivery to the prostate or periprostate space or transrectal injection can be used.
In the context of radioimmunotherapy, the conjugates of the present invention are introduced into the body and are allowed to concentrate in the target region. The therapeutic effect occurs when the conjugates are near or in contact with and bind to the targeted cells. Cell death can be a direct or indirect result of the radiation event of the radiometal that is positioned in close proximity to the cell. Desirably, the conjugate comprises a monoclonal antibody that is specific (e.g., for a cell-surface molecule) for a cell, such as a diseased cell, to be killed. Cell death is caused by decay of the radiometal and can occur in one of two ways. First, if the alpha particle is emitted in the direction of the diseased cell, a single hit in the cell nucleus can be cytotoxic. The isotope to which the radiometal decays after emitting the alpha particle is ejected from the chelate on a trajectory opposite that of the alpha particle. The bound cell, therefore, can still be hit even when the alpha particle is emitted on a trajectory away from the cell. A single hit in the cell membrane by the decayed isotope can cause irreparable cell injury leading to cell death. The relatively high effectiveness of the alpha particle means that less radioactive material can be employed. Selectivity of the targeting agent, e.g., monoclonal antibody, and the short range (a few cell diameters) of the alpha particles minimizes the destruction of healthy tissue on a cellular level.
The benefits that inure to this embodiment of the invention are numerous. The high specificity of the conjugate minimizes total radiation dosage. Only enough radiation for the target cells need be employed. In addition, 225Ac-HEHA is cleared rapidly from the body should the targeting agent be disrupted. Additionally, since the amount of radiometal employed is minimized, the radiation hazard to persons preparing and administering the conjugate is significantly reduced. In addition, tissue damage or whole body dose during therapy is markedly reduced as compared to that from presently employed methods of radiation therapy, such as isotope implants, external radiation therapy, and immunoradiotherapy employing iodine-131 labeled polyclonal or autologous antibodies. Additionally, both biological and physical half-lives of the targeting radiobiological can now be controlled, minimizing whole body radiation effects. Since radiation is targeted to specific cell types, such as neoplastic cells, a therapeutic dose is delivered specifically to malignant cells, either localized or metastasized. The ability of conjugates to provide an effective dose of therapeutic radiation specifically to metastasized cells is also unique and singularly useful for cancer therapy. Desirably, when the cancer is a solid tumor, 225Ac-HEHA or 225Ac-HEHA-targeting agent is administered intratumorally. Any leakage of decay products to the surrounding healthy tissue can be chelated by the subsequent or simultaneous administration of HEHA or the like. Desirably, a 225Ac-HEHA-targeting agent that targets leukemic cells or prostate cancer cells is administered in the treatment of leukemia or prostate cancer, respectively. The 225Ac-HEHA-targeting agent desirably is internalized by the cell and all decay occurs within the cell, preferably with the daughter isotopes decaying thereafter within one hour or less, thereby minimizing any damage to normal cells and tissue.
In view of the above, the present invention provides a method of treating disease. The method comprises administering to a patient having disease a disease-treatment effective amount of 225Ac-HEHA-targeting agent in which the targeting agent is specific for diseased cells. Preferably, the targeting agent is an antibody.
Also provided is a method of treating cancer. The method comprises administering to a patient having cancer a cancer-treatment effective amount of 225Ac-HEHA-targeting agent in which the targeting agent is specific for the cancer. Preferably, the targeting agent is an antibody. If the cancer is a solid tumor, the method preferably comprises intratumorally administering to a patient having a tumor a tumor-treatment effective amount of 225Ac-HEHA or 225Ac-HEHA-targeting agent, in which the targeting agent is specific for the tumor. Any leakage of decay products to the surrounding healthy tissue can be chelated by the subsequent or simultaneous administration of HEHA or the like.
A method of decontaminating a sample from 225Ac is also provided. The method comprises contacting the sample with a decontaminating-effective amount of HEHA. A decontaminating-effective amount can be determined in accordance with methods known in the art. Preferably, the HEHA is attached to a solid support and the sample is a liquid. This method has application in the context of bioremediation.
Further provided is a method of decontaminating a person who has been externally contaminated with 225Ac. The method comprises contacting the person with a decontaminating-effective amount of HEHA. A decontaminating-effective amount can be determined in accordance with methods known in the art. Similarly, a method of detoxifying a person who has internalized 225Ac is also provided. The method comprises administering to the person a detoxifying-effective amount of HEHA. A detoxifying-effective amount can be determined in accordance with methods known in the art.